Project Summary/Abstract Leptospira interrogans is maintained in a zoonotic cycle in which rats are the primary reservoir. Infection begins when a reservoir or an incidental host, including humans, comes into contact with water or soil contaminated with urine from animal reservoirs. Following acquisition, leptospires switch from a free-living to a parasitic life-style, a complex, multifactorial process often referred to as mammalian host adaptation. The outcome of these early events differ dramatically depending on whether infection occurs in a reservoir- competent or -incompetent (i.e., incidental) host. In reservoir hosts, leptospires are cleared within days from all sites except the kidney, where they colonize the proximal renal tubules. In contrast, hamsters and guinea pigs, the species most commonly used to study acute leptospirosis, are exquisitely sensitive to infection, succumbing to disease with very low inocula. These observations imply that the outcome following infection is determined largely by the nature and intensity of the host?s inflammatory immune response. Thus, a key premise underlying our proposal is that Leptospira use highly similar, if not identical, genetic programs to establish themselves within asymptomatic (i.e., reservoir) and symptomatic (i.e., incidental) hosts despite the markedly divergent immunopathological events following infection. To study the genetic pathways and genes induced by Leptospira in response to in vivo stimuli, we developed a novel animal model in which leptospires are cultivated within dialysis membrane chambers (DMCs) implanted within the peritoneal cavities of rats, the natural reservoir. Using the DMC model in conjunction with RNAseq, we identified >160 genes that are differentially expressed by L. interrogans in response to mammalian host-specific signals. These transcriptomic data led us to hypothesize that environmental sensing within the DMC milieu triggers genetic programs required by Leptospira to infect/colonize the mammalian host. The transposon mutants screening approach outlined in Aim 1 will enable us to identify individual leptospiral gene products involved in environmental sensing, physiological adaptation and virulence during mammalian infection. To understand the genetic basis for renal colonization, in Aim 2, we will examine the expression profile of Leptospira within the kidneys of infected rats.